Method for the production of nuclear transfer embryos

ABSTRACT

Methods for the production of nuclear transfer embryos, nuclear transfer embryos and animals derived therefrom are described. The method generally comprises at least the steps of: providing at least one enucleated recipient cell; providing at least one donor cell or nucleus; providing a fusion media which is substantially free of calcium; placing said at least one enculeated recipient cell and at least one donor cell or nucleus in contact with one another to form couplets; and, fusing via electrofusion in said fusion media said at least one recipient cell with at least one donor cell or nucleus to form a nuclear transfer embryo.

FIELD

[0001] The present invention relates to methods for the production ofnuclear transfer embryos. The methods are particularly beneficial to theproduction of porcine nuclear transfer embryos. More particularly, themethods allow for fusion of donor and recipient cells to form nucleartransfer embryos prior to activation thereof.

BACKGROUND

[0002] Nuclear transfer involves insertion of a nucleus or whole nucleardonor cell (karyoplast) into an enucleated oocyte (cytoplast orrecipient cell) followed by fusion of the karyoplast and cytoplast toform a single cell nuclear transfer (NT) embryo. Fusion results in thereprogramming of the donor nucleus by the recipient cytoplasm. Uponsuitable activation cleavage division and development may be initiated.Accordingly, an activated single cell NT embryo is a viable embryo,capable of cell division to give a multicellular activated embryo, whichis competent to develop in culture to a blastocyst stage.

[0003] Activated nuclear transfer embryos may be introduced into theuterus of a synchronised recipient animal, for example, after culture tothe blastocyst stage, to give cloned animals.

[0004] Nuclear transfer or cloning using somatic cells has beensuccessfully performed in a variety of animals such as cattle (Cibelliet al 1998 Science 280:1256) and sheep (Wilmut et al (1997) Nature385:810).

[0005] A number of standard nuclear transfer techniques employed inspecies such as cattle and sheep involve electrofusion. When employed inporcine cells, utilizing standard fusion media, such a technique oftenresults in concurrent activation of the recipient cytoplast. Suchactivation is undesirable at such stage of the procedure. For example,activation induces a large decrease in the levels of maturationpromoting factor (MPF) activity in oocytes, high levels of which aregenerally associated with reprogramming the donor nucleus followingfusion. Accordingly, premature activation may interfere with the abilityof the cytoplast to reprogramme the donor nucleus leading to decreaseddevelopmental competence of the embryo. Premature activation may alsotrigger other cellular events, such as (pro)nucleus formation, beforereprogramming of the donor nucleus was complete.

[0006] It is considered that avoiding simultaneous fusion and activationof nuclear transfer embryos may have the advantage of providing thenuclear transfer procedure with flexibility in the type of activationtreatment that may subsequently be utilised.

[0007] The inventors of the present invention have identified that ifelectrofusion is conducted using media substantially free of calcium theproblem of simultaneous fusion and activation of at least porcinederived NT embryos may be overcome.

[0008] Further the inventors of the present invention have surprisinglydiscovered that in certain cases holding or incubating couplets in mediasubstantially free of calcium for a period prior to electrofusion and NTembryos in a media substantially free of calcium for a period followingelectrofusion may further help overcome the problem of prematureactivation of at least porcine derived NT embryos. The inventor's havefound this to be particularly applicable where in vitro matured (IVM)oocytes are utilised as cytoplasts.

[0009] Accordingly, the invention described herein provides an efficientmeans of producing at least porcine authentic nuclear transfer embryos.

STATEMENT OF INVENTION

[0010] In one aspect, the present invention provides a method for theproduction of nuclear transfer embryos comprising at least the steps of:

[0011] providing at least one enucleated recipient cell;

[0012] providing at least one donor cell or nucleus;

[0013] placing said at least one enucleated recipient cell and at leastone donor cell or nucleus in contact with one another to form couplets;

[0014] providing a fusion media which is substantially free of calcium;and

[0015] fusing via electrofusion, in said fusion media, said at least onerecipient cell with at least one donor cell or nucleus to form a nucleartransfer embryo.

[0016] Preferably, said recipient and donor cells used in a method ofthe invention are porcine.

[0017] Preferably, the parameters of said electrofusion are a singleelectrical pulse at between 100V to 200V for between 30 μs and 100 μsover an electrode gap of 1 mm. More preferably, the parameters of saidelectrofusion are a single electrical pulse at 1.5 kV/cm for 60 μs.

[0018] Preferably, said recipient cell is a freshly ovulated or afollicular oocyte arrested at MII. Alternatively, said recipient cell isan in vitro-matured (IVM) oocyte.

[0019] Preferably, the couplets are held in media which is substantiallyfree of calcium for a period prior to electrofusion to form a nucleartransfer embryo. Preferably the period is at least approximately 15minutes.

[0020] Preferably, a method of the invention further comprises the stepof incubation of NT embryos in a media which is substantially free ofcalcium for a period following electrofusion. Preferably the period isat least approximately 15 minutes.

[0021] Preferably the donor cell or nucleus used in a method of theinvention is a somatic cell. More preferably said donor cell is afibroblast.

[0022] In another aspect, the present invention provides a method ofcloning animals comprising at least the steps of:

[0023] producing a nuclear transfer embryo according to a methodhereinbefore described;

[0024] activating said nuclear transfer embryo to provide an activatedembryo;

[0025] optionally allowing said activated embryo to undergo at least oneround of cell division;

[0026] transferring activated and divided embryo to a synchronisedfemale recipient animal;

[0027] allowing said synchronised female recipient animal to carry saidembryo to full gestation to produce a cloned animal.

[0028] Preferably, said nuclear transfer embryo is porcine and saidrecipient female animal and said cloned animal are pigs.

[0029] Preferably, where the nuclear transfer embryo is constructedusing in vivo-derived oocytes, said nuclear transfer embryo is held in acalcium-containing media with serum prior to activation thereof.

[0030] Preferably, where the nuclear transfer embryo is constructedusing in vitro-matured (IVM) oocytes, said nuclear transfer embryo isheld in a media substantially free of calcium for a period prior toactivation thereof. Preferably, said period is at least 15 minutes.

[0031] Preferably, said nuclear transfer embryos are activated no laterthan 5 hours post fusion.

[0032] In another aspect, the present invention provides a method forthe production of porcine nuclear transfer embryos comprising at leastthe steps of:

[0033] providing at least one enucleated recipient cell of porcineorigin;

[0034] providing at least one donor cell or nucleus of porcine origin;

[0035] placing said at least one enucleated recipient cell and at leastone donor cell or nucleus in contact with one another to form couplets;

[0036] optionally providing a first media which is substantially free ofcalcium;

[0037] optionally incubating said couplets in said first media for aperiod of preferably at least approximately 15 minutes;

[0038] providing a second media which is substantially free of calcium;

[0039] fusing via electrofusion, in said second media, said at least onerecipient cell with at least one donor cell or nucleus to form a nucleartransfer embryo;

[0040] providing a third media which is substantially free of calcium;and

[0041] incubating said nuclear transfer embryo in said third media for aperiod preferably of at least approximately 15 minutes followingelectrofusion.

[0042] Preferably, the recipient cell is derived from in vitro maturedoocytes.

[0043] In another aspect, the present invention provides a method ofcloning pigs comprising at least the steps of:

[0044] producing a porcine nuclear transfer embryo according to themethod of the two immediately preceding paragraphs herein;

[0045] activating said nuclear transfer embryo to provide an activatedembryo;

[0046] optionally allowing said activated embryo to undergo at least oneround of cell division;

[0047] transferring activated and divided embryo to a synchronisedfemale recipient animal;

[0048] allowing said synchronised female recipient animal to carry saidembryo to full gestation to produce a cloned pig.

[0049] The invention may also be said broadly to consist in the parts,elements and features referred to or indicated in the specification ofthe application, individually or collectively, in any or allcombinations of two or more of said parts, elements or features, andwhere specific integers are mentioned herein which have knownequivalents in the art to which the invention relates, such knownequivalents are deemed to be incorporated herein as if individually setforth.

PREFERRED EMBODIMENT(S)

[0050] The following is a description of the preferred forms of thepresent invention given in general terms. The invention will be furtherelucidated from the non-limiting Examples provided hereinafter.

[0051] The present invention generally relates to the production ofnuclear transfer embryos, particularly porcine nuclear transfer embryos,utilising electrofusion of a recipient cell (cytoplast), and a donornucleus or donor cell (karyoplast). The inventors have identified thatat least in porcine cells if such electrofusion is conducted in mediasubstantially free of calcium that undesirable concurrent activiation ofthe nuclear transfer embryo will not occur. Further, the inventors havesurprisingly found that if couplets in accordance with the invention areheld in a media substantially free of calcium for a period prior toelectrofusion and formed nuclear transfer embryos held in such media fora period post electrofusion, that this may further help preventundesirable premature activiation of the nuclear transfer embryo,particularly in the case of porcine nuclear transfer embryos. Thisembodiment is particularly applicable where in vitro matured (IVM)oocytes are used as cytoplasts.

[0052] As used herein “substantially free of calcium” should be taken tomean that the media referred to is free of a level of calcium that mayact to induce or stimulate activation of the embryo such that cleavagedivision and development may be initiated; ie such that the embryo iscompetent to develop to the blastocyst stage. Preferably, the mediacontains no calcium.

[0053] The recipient cytoplast can be derived from an oocyte, zygote orany cell from an embryo. Ova or oocytes may be readily collected fromthe reproductive tracts of ovulating animals using surgical ornon-surgical methods. Methods for isolating oocytes are well known inthe art. Ovulation may be induced by administering gonadotropins ofvarious species origin to animals. Oocytes may be collected byaspiration from mature follicles, or collected following ovulation.Alternatively immature oocytes may be collected from the ovaries ofliving or slaughtered animals and matured in vitro (IVM oocytes) usingstandard procedures such as described in WO 90/13627 (“In vitromaturation of bovine oocytes in media containing recombinantgonadotropins along with bovine oviductal cells”, 1989). Oocytes can befertilised in vivo or in vitro to yield zygotes.

[0054] Accordingly to one embodiment of the present invention thecytoplast is derived from an ovulated unfertilised oocyte. Morepreferably, the oocyte is a freshly ovulated (less than 12 hours postovulation) or follicular oocyte and is arrested in metaphase of thesecond meiotic division (MII). However, those of general skill in theart to which the invention relates will appreciate that a cytoplast maybe derived from fertilized oocytes, embryo blastomeres, embryonic stemcells, primordial germ cells and somatic cells.

[0055] Accordingly to a particularly preferred embodiment of theinvention as will be described herein the cytoplast is an IVM oocytecollected from the ovaries of living or slaughtered animals and maturedin vitro using standard procedures.

[0056] Formation of the cytoplast via enucleation may occur according tothe invention by any one of a number of standard techniques used in theart; for example, bisection of an oocyte, enucleation of the metaphaseplate, self enucleation. The procedure elucidated in Example 1 hereinprovides one detailed example.

[0057] It will appreciated that the methods of the present invention areparticularly directed at pigs. However, the inventors believe themethods likely to be applicable to any species of animal, includinglivestock animals and companion animals. Accordingly, the recipientcytoplast may come from any such animal.

[0058] The donor nucleus or cell (either being referred to herein as akaryoplast) may be derived from any type of somatic cell, be they foetalor adult, including embryonic stem cells. The cells may be derived fromfresh tissue samples or alternatively from cultured cell lines.Preferably, fibroblast cells are used as karyocytes. Fibroblasts areeasily obtained (either from foetal or adult tissue sources), can beobtained in large quantities and are easily propagated, geneticallymodified and cultured in vitro.

[0059] The invention particularly relates to donor nucleus or karyoplastderived from pigs. However, methods of the invention may be applicableto other animals. Accordingly the karyoplast may come from any animalincluding livestock animals or companion animals. It will be appreciatedthat a donor nucleus or karyoplast derived from an animal can beisolated from any appropriate type of tissue or organ. As it will beappreciated, the karyoplast is preferrably derived from a species ofanimal equivalent to that from which the cytoplast has been derived.

[0060] The importance of synchronising the cell cycle between the oocyte(cytoplast) and the donor nucleus has been demonstrated previously. Highlevels of maturation promoting activity in the metaphase II oocyteresult in irreversible damage to the chromatin and aneuploid followingreconstruction (Campbell et al 1993 Biology of Reproduction 49:933). Toovercome this problem the cell cycle of the karyoplast needs to be inmetaphase or G1 of the cell cycle. Donor nuclei can have the cell cyclesynchronised using a variety of methods such as serum starvation (Wilmutet al 1997), growth to confluence (Onishi et al 2000), etc.Non-synchronised populations can also be used (Cibelli et al 1998).Alternatively the oocyte or recipient (cytoplast) can be activated toreduce MPF levels (so called universal recipient).

[0061] Following preparation of each of the cytoplast and karyoplast thecells are placed in contact with one another, such that the cytoplasm ofthe cytoplast comes into contact with that of the karyoplast, to formwhat may be called cell “couplets”. Such contact may be establishedaccording to known techniques; examples of such techniques are providedin Examples 1 and 2 herein.

[0062] Following formation the “couplets” may be held in a suitablemedia for a period prior to fusion as herein after described. Suchperiod may be between approximately 15 minutes to 3 hours. Such suitablemedia may include any suitable holding or cell culture media, as will berecognised by persons of general skill in the art to which the inventionrelates.

[0063] The inventors have identified that in at least the case where IVMooctyes have been utilised as cytoplasts, the couplets, while they mayhave been formed in a media containing calcium, are preferablytransferred to, or maintained in, a media substantially free of calciumfor a period prior to fusion. Preferably said period is at leastapproximately 15 minutes.

[0064] As used herein “prior to fusion” should be taken to mean justprior to, or immediately prior to, the commencement of the electrofusionprocess. It will be appreciated that the word “immediately” is used in abroad sense and should not be taken to mean that no time has lapsedbetween the end of said period and electrofusion. As will beappreciated, in certain instances a short amount of time may lapsebetween the end of said period and the electrofusion process on thebasis of the time it may take to manipulate and prepare a sample forfusion, for example, by transferring the couplets from a suitableholding or cell culture media to a suitable electrofusion media.However, it will be appreciated that no intervening steps are to occurin which the couplets are placed in a calcium-containing media.

[0065] Suitable media substantially free of calcium for incubation ofcouplets for a period prior to fusion will readily be recognised bypersons of ordinary skill in the art to which the invention relates andmay include any suitable known suitable holding or cell culture media,for example as herein after described. A preferred media is calcium freepNCSU-23 as hereinafter described. The inventors also envisage asituation where a fusion media may represent such suitable media. Inthis situation, the fusion media is best adapted such that extendedincubation therein will not substantially degrade the couplets or reduceviability thereof.

[0066] In the case of the use of freshly ovulated or follicular oocytesarrested at MII the inventors have found that couplets may be held in amedia containing calcium up until fusion is to occur. However, theinventors believe that incubation for a period in media substantiallyfree of calcium for a period prior to fusion, as mentioned in thepreceding paragraph, may be beneficial, or at least will not bedetrimental, to formation of NT embryos in this instance.

[0067] Permanent transfer of the donor nucleus into the recipientcytoplast, or fusion, according to the invention, is effected byelectrofusion. Electrofusion may occur in any commonly available fusionmachine; for example a BTX Electro-Cell-Manipulator ECM 2001 (BTX, Inc).Generally, groups of couplets, preferably comprising 10 or less coupletsper group, are suspended in any suitable known electrofusion media whichis substantially free of calcium, and placed in a fusion chamber of thefusion machine, for electrofusion. Examples of such media includecalcium-free Zimmerman's medium, and calcium-free Mannitol fusion mediaas described in the Examples which follow.

[0068] Electrofusion according to the invention preferably involves thedelivery of a single electrical pulse at 1.5 kV/cm by the fusion machineto the nuclear transfer couplets. The pulse is preferably delivered fora duration of 60 microseconds over an electrode gap of 1 mm. Theinventors have found that utilising these parameters, in combinationwith employing a fusion media which is substantially free of calcium,fusion may occur without activation of the cytoplast. While theinventors believe the above parameters are preferable, they haveidentified that fusion without activation may be achieved using a pulsefield strength from between 100V to 200V and a pulse duration from 30 to100 microseconds, with an electrode gap of 1 mm.

[0069] The inventors believe it important that only a single DC pulse bedelivered to obtain fusion without activation. However, they haveidentified that couplets which remain unfused 0.5 h after application ofa fusion pulse according to the invention may be exposed to a secondsuch fusion pulse and undergo fusion without activation.

[0070] Following fusion and prior to activation according to theinvention, the nuclear transfer embryos may be transferred to a suitableholding or cell culture media and maintained in a viable state undersuitable conditions, for example at 39° C. and 5% CO₂. While certainconditions and culture media are exemplified herein after, those skilledin the art will readily appreciate alternative conditions and culturemedia which may be employed to maintain the embryos in a viable statesuch that they may be subsequently activated to divide and develop.

[0071] The inventors have identified that at least in the case where IVMoocytes are utilised as cytoplasts it is particularly preferable thatthe NT embryos be maintained or transferred to a media substantiallyfree of calcium for a period post fusion as herein before described.Preferably, this period is at least approximately 15 minutes. Followingthis initial period, NT embryos may be transferred to a calciumcontaining media to be held prior to activation.

[0072] As used herein “a period post fusion” should be taken to meanjust following, or immediately following, the completion of theelectrofusion process. It will be appreciated that the word“immediately” is used in a broad sense and should not be taken to meanthat no time has elapsed between the end of the electrofusion processand the beginning of said period. As will be appreciated, in certaininstances a short amount of time may lapse between the completion ofelectrofusion and the beginning of said period on the basis of the timeit may take to manipulate NT embryos post fusion, for example, bytransferring NT embryos to a suitable holding or cell culture mediasubstantially free of calcium.

[0073] Suitable media substantially free of calcium to be utilised inaccordance with this aspect of the invention will be readily recognisedby persons skilled in the technological field of the invention, andpreferably represent a suitable holding or cell culture media One suchsuitable media substantially free of calcium is exemplified in Example 2herein after; calcium free pNCSU-23. The inventors also envisage asituation where the media in which electrofusion took place representsthe suitable media substantially free of calcium. In this instance, thefusion media is best adapted not to substantially degrade the NT embryosor reduce viability thereof.

[0074] In the case where freshly ovulated or follicular oocytes arrestedat MII are utilised as cytoplasts the inventors have identified thatfollowing electrofusion NT embryos may be transferred to and maintainedin a suitable holding or culture media containing calcium (for exampleNCSU 23 supplemented with 10% FCS). However, the inventors believe thatincubation for a period in media substantially free of calcium followingelectrofusion, as mentioned in the preceding paragraph, may bebeneficial, or at least will not be detrimental, to the viability of theNT embryos and to the end of preventing premature activation, in thisinstance.

[0075] While the inventors believe it to be particularly preferable, atleast in the case where IVM oocytes are used as cytoplasts, that bothpre and post fusion incubation for a period in media substantially freeof calcium (as herein before described) be effected, they envisage thatthe pre-fusion incubation of couplets in media substantially free ofcalcium may be omitted while still maintaining viability of derived NTembryos and preventing premature activation thereof. Accordingly, thepresent invention will be understood to encompass methods in whichcouplets are transferred from a calcium containing culture or holdingmedia to a fusion media substantially free of calcium, electrofusion isconducted, and the resultant NT embryos subsequently transferred to ormaintained in a suitable media substantially free of calcium for aperiod in accordance with the invention.

[0076] Following formation of the nuclear transfer embryos according tothe invention the embryos may be activated such that cleavage divisionand development is initiated. Such activation may occur by any meanscurrently known in the art. For example, in relation to the activationof porcine oocytes or embryos the method reported by Önishi et al(Science, 289:1188-1190 (2000)) may be utilised. Other activationtechniques that may be utilized include the application of electricalpulses (in calcium-containing medium) and incubation with chemicalreagents, such as calcium ionophores, ethanol or thimerosal. Further,preferable means of activation of porcine-derived nuclear transferembryos are detailed in Examples 1 and 2 herein.

[0077] The inventors believe that in order to maintain viability andcompetency of the nuclear transfer embryos prepared according to theinvention the embryos should preferably be activated no later than 5hours post fusion, preferably no later than 3 hours post fusion. Bydoing so, the inventors believe that DNA fragmentation of the karyoplastchromosomes may be substantially prevented.

[0078] Following activation, the NT embryos may be cultured in vitro forone or more divisions. After cleavage, the NT embryos may be bisected atany suitable stage, (for example, at the 2 to 32 cell stage) usingphysical or chemical means (embryo splitting). Embryonic cells orblastomeres may be isolated therefrom and used in second and subsequentrounds of nuclear transfer to produce multiple NT embryos capable ofdevelopment to term (serial cloning).

[0079] A second round of nuclear transfer has been used to increase thedevelopmental competence of mouse NT embryos (Kwon & Kono (1996) Proc.Natl. Acad. Sci. USA 93:13010) and the inventors contemplate thesuitability of this technique in combination with the present invention.The second cytoplast can be an oocyte, zygote or any other embryo.

[0080] It will be appreciated that NT embryos produced according to theinvention can be cultured in vitro for one or more divisions to assesstheir viability or transferred to the reproductive tract of a recipientfemale animal, or stored frozen for subsequent use by standardprocedures.

[0081] The present invention may include the genetic manipulation of theDNA of the donor nucleus or karyoplast prior to transfer into therecipient cytoplast. Alternatively, or in addition, genetic manipulationmay take place following NT cell production, that is geneticmanipulation on the NT embryo is contemplated.

[0082] Where activated NT embryos produced according to the inventionare to be used for production of cloned animals, the embryo istransferred to the reproductive tract of a synchronised recipientfemale. As used herein “synchronised recipient” should be taken to meana suitable female animal whose oestrus cycle has been synchronised suchthat its uterus is ready to accept an NT embryo and carry it to fullgestation. It will be appreciated that a recipient female animal may besynchronised according to commonly utilised methods such as the hormonaltreatment of mated and aborted gilts.

[0083] Uses for nuclear transfer or cloning technology according to thepresent invention include: the production of genetically identical orsimilar animals or clones from an individual animal for purposes ofanimal breeding; the production of genetically manipulated, that is,transgenic animals in which extra genetic information has been insertedor existing genetic information deleted (gene knockout); and thede-differentiation of somatic cells to produce a population ofpluripotent cells which can then be differentiated to cells, tissues ororgans for the purpose of cell therapy, gene therapy, organtransplantation, etc. Such cells have an advantage in that they can beautologous, that is, obtained initially from the patient and as such arenot destroyed by the patient's immune system.

[0084] It will be appreciated that “animals” as used herein may belivestock or companion animals. Preferably, the animal is a mammal andmore preferably a pig.

[0085] Thus, according to the present invention, reproduction ormultiplication of animals, preferably mammals, particularly pigs, havingspecific or desired genotypes is possible. In addition, the presentinvention can also be used to produce animals which can be used, forexample, in cell, tissue or organ transplantation, or to produce animalswhich express desired compounds such as therapeutic molecules, growthfactors, or other medically desired peptide or protein.

EXAMPLES Example 1

[0086] Production of Cloned Pigs Using In Vivo Oocytes as Cytoplasts andUsing Fusion Before Activation

[0087] Materials:

[0088] Dulbecco's Phosphate Buffered Saline (DPBS; 136.98 mM NaCl, 2.68mM KCl, 0.49 mM MgCl₂.6H₂O, 8.08 mM Na₂HPO₄, 1.47 mM KH₂PO₄ and 0.90 mMCaCl₂.2H₂O; pH 7.4) supplemented with 1% Foetal Calf Serum (FCS)

[0089] Hepes buffered MEM, consisted of Minimum Essential Medium (withEarle's salts, L-glutamine and non-essential amino acids; Gibco-BRL,Grand Island, N.Y., USA) supplemented with 336 mg/L NaHCO₃, 21 mM Hepesbuffer, 60 mg/L penicillin-G and 0.5% Bovine Serum Albumin (BSA).

[0090] pNCSU-23 (127.8 mM NaCl, 4.97 mM KCl, 1.0 mM KH₂PO₄, 1.19 mMMgSO₄.7H₂O, 3.0 mM Na₂HPO₄, 5.55 mM D-glucose, 75 mg/L penicillin-G, 50mg/L streptomycin sulfate, 1.7 mM CaCl₂, 1.0 mM L-glutamine, 7.0 mMtaurine, 5.0 mM hypotaurine, 0.4% BSA and 10% FCS)

[0091] NCSU-23 (108.73 mM NaCl, 4.78 mM KCl, 1.19 mM KH₂PO₄, 1.19 mMMgSO₄.7H₂O, 5.55 mM D-glucose, 75 mg/L penicillin-G, 50 mg/Lstreptomycin sulfate, 1.7 mM CaCl₂, 1.0 mM L-glutamine, 7.0 mM taurine,5.0 mM hypotaurine and 0.4% BSA)

[0092] Dulbecco's Modified Eagle Medium (DMEM; high glucose withL-glutamine, 110 mg/L sodium pyruvate and pyridoxine hydrochloride:Gibco-BRL)

[0093] Calcium-free mannitol fusion medium (0.28 M mannitol, 0.2 mMMgSO₄ and 0.01% polyvinylalcohol)

[0094] Modified TALP-PVA medium (114.0 mM NaCl, 3.16 mM KCl, 0.35 mMNaH₂PO₄.2H₂O, 0.5 mM MgSO₄.6H₂O, 25 mM NaHCO₃, 2 mg/L phenol red, 0.1%PVA, 75 mg/L penicillin-G, 50 mg/L streptomycin sulfate, 4.72 mMCaCl₂.2H₂O, 10.0 mM sodium lactate, 0.10 mM sodium pyruvate, 2.0 mMcaffeine-sodium benzoate, 3.0 m calcium lactate and 0.4% BSA)

[0095] Method:

[0096] Freshly ovulated oocytes were flushed using Dulbecco's PhosphateBuffered Saline (DPBS) from superstimulated pig donors 48 h after hCGinjection and transported to the laboratory in Hepes buffered MEM. Theywere then stripped from their remaining cumulus by pipetting in pNCSU 23containing 10 mg/ml hyaluronidase. Stripped oocytes were then washed andcultured in NCSU 23 with 10% FCS at 5% CO₂, 39° C. for 0.5-2 h prior tomicromanipulation.

[0097] Primary cultures of porcine foetal fibroblast cells grown toconfluency after 7-14 days culture in DMEM with 15% FCS at 5% CO₂, 39°C. were used as karyoplasts. They were prepared for nuclear transfer bywashing confluent monolayers twice with DPBS followed by the addition ofDPBS containing 0.05% trypsin. After 5 minutes of incubation at 39° C.,DMEM+15% FCS was added to dissociated cells to stop the trypsinreaction. Dissociated cells were then pelleted by centrifugation at300×g for 5 minutes and resuspended in DMEM+15% FCS. Dissociated cellswere incubated at 5% CO₂, 39° C. for at least 0.5 h prior tomicromanipulation.

[0098] For micromanipulation, oocytes and cells were placed in a dropunder oil of pNCSU 23 with 7.5 μg/ml cytochalasin B and 10% FCS. Oocyteswere enucleated by removing the first polar body along with adjacentcytoplasm containing the metaphase plate using a micropipette with aninner diameter of about 20 μm. In a majority of oocytes, the metaphaseplate was visible under phase contrast optics as a clear spacecontrasted against dark cytoplasm. Through the same hole in the zonapellucida created during enucleation, a small to medium-sized cell wasthen placed in contact with the cytoplasm of each oocyte to create acouplet. After manipulation, couplets were washed once and cultured inNCSU 23 with 10% FCS at 39° C., 5% CO₂ in air for at least 0.5 h beforefusion.

[0099] Just prior to fusion, couplets were removed from the incubatorand placed in drops of pNCSU 23 under oil. Groups of up to 10 coupletswere washed thoroughly in calcium-free Mannitol fusion medium and thenimmediately transferred to a fusion chamber with two electrodes 1 mmapart overlaid with fusion medium. Couplets were manually aligned usinga 30 gauge needle so that the plane of contact between the donor andrecipient cells was parallel with the electrodes. Cell fusion wasinduced with a single DC pulse of 150 V/mm for 60 μsec. Couplets werealso exposed to a 4.0V AC pulse for 2 sec immediately prior to thefusion pulse and to an AC pulse immediately after the fusion pulse, thatdiminished from 4.0 to 0.0V over a 2 sec interval. After electricalpulse, couplets were returned to pNCSU 23 drops for at least 0.5 h.Unfused couplets were exposed to the same fusion procedure a secondtime. Fused embryos were returned to the incubator at 5% CO₂, 39° C. inNCSU 23 with 10% FCS for 1-3 h prior to activation.

[0100] Activation of reconstructed porcine zygotes was conducted usingthe ionomycin/6-DMAP (6-dimethylaminopurine) method describedhereinafter. One to 3 h post fusion, the fused couplets (or single cellnuclear transfer embryos) were placed in modified TALP-PVA mediumsupplemented with 3.0 mM Ca-lactate (mTALP-PVA) for approximately 0.5hour prior to activation. Fused couplets were then transferred tomTALP-PVA containing 5 μM ionomycin for five minutes. Fused coupletswere then washed twice and incubated in culture medium (NCSU 23+0.4%BSA) containing 2 mM 6-dimethylaminopurine (6-DMAP) for three hours.Activated fused couplets were then washed twice and transferred to 50 μldroplets of the culture medium under mineral oil and cultured for either7 days to assess in vitro development or for 3 days prior to transferinto a synchronised recipient.

[0101] Results:

[0102] Results obtained from the method of Example 1 are outlined inTables 1-3. The results demonstrate that the embryo reconstructionprotocol detailed herein achieves fusion of the donor cell without theconcomitant activation of the recipient cytoplasm.

[0103] The data presented in Table 1 shows development of couplets fusedin media with or without calcium using the fusion method as describedherein without subsequent activation using ionomycin/DMAP. Asdemonstrated in Table 1, the vast majority of couplets fused in calciumfree medium did not cleave after 48 h. This indicates that donor cellswere successfully fused without activating the recipient cytoplasts.TABLE 1 Development of fused couplets after two days of culture whendonor cells were fused in the presence or absence of calcium¹. CleavedLysed/fragmented Fusion treatment No. cybrids (%)³ (%)³ Calcium present54 37 (69)^(a) 4 (7)  Calcium absent 73  7 (10)^(b) 8 (11)

[0104] Embryos reconstructed using the fusion before activation protocoloutlined herein were capable of development to blastocyst stage at highefficiency. From 108 reconstructed embryos that were subsequentlyactivated using ionomycin/6-DMAP, 25 developed to hatching blastocyststage after 6 days in culture (Table 2). To verify that fusion occurredwithout activation, control fused couplets were also exposed to thefusion conditions and subsequently cultured. The majority of the fusedcouplets obtained according to Example 1 remained uncleaved, clearlyindicating that concomitant activation in most of the recipientcytoplasts did not occur during fusion. Differential staining of theseembryos showed that nuclear transfer blastocysts after day 6 had goodnumbers of inner cell mass (average 10) and trophectoderm cells (average31). TABLE 2 Development of fused couplets with or without activationusing ionomycin followed by incubation in 6-DMAP¹. Cleaved BlastocystTreatment No. cybrids (%)² (%)² Activated 108 100 (93)^(a) 25 (23)^(a)Not activated 27  2 (7)^(b) 0 (0)^(b)

[0105] The efficiency of the pig cloning procedure outlined in thisexample is also observed when reconstructed embryos were cultured for 3days then subsequently transferred to synchronised recipients (Table 3).An average of 70% of couplets manipulated were successfully fused ofwhich 90% cleaved and were transferred to recipients. From 5 transfers,2 recipients were found to be pregnant at day 40 giving a high pregnancyinitiation of 40%. These recipients subsequently farrowed one livecloned piglet each. TABLE 3 Development of NT porcine embryos lysed/pregnancy status day couplets fused uncleaved 2-cell 3-cell 4-cell5-cell+ fragment (day 40) cloned pigs 5/12/000 99 64 8 7 5 26 13 5pregnant 1 14/12/00 108 49 2 8 4 13 16 6 not pregnant 19/12/00 103 77 720 13 24 5 8 not pregnant 2/1/01 106 96 1 13 9 44 29 0 not pregnant4/1/01 148 110 3 16 20 47 23 1 pregnant 1 Average 113 79 4 13 10 31 17 42/5

Example 2

[0106] Creation of Cloned Porcine Embryos Using In Vitro Matured Oocytesas Recipient Cytoplasts

[0107] Materials:

[0108] Calcium-free (Ca-free) pNCSU-23 (127.8 mM NaCl, 4.97 mM KCl, 1.0mM KH₂PO₄, 1.19 mM MgSO₄.7H₂O, 3.0 mM Na₂HPO₄, 5.55 mM D-glucose, 75mg/L penicillin-G, 50 mg/L streptomycin sulfate, 1.0 mM L-glutamine, 7.0mM taurine, 5.0 mM hypotaurine, 0.4% BSA and 10% FCS)

[0109] Oocyte maturation medium (OMM199a) Medium 199 (with Earle'ssalts, L-glutamine, 2.2 mg/ml sodium bicarbonate and 25 mM Hepes buffer;Gibco-BRL Grand Island, N.Y., USA) supplemented with 0.1 mg/ml sodiumpyruvate, 75 μg/ml penicillin-G, 50 μg/ml streptomycin sulfate, 10 μg/mlovine FSH, 5.0 μg/ml ovine LH, 1.0 μg/ml 17β-estradiol, 0.5 mMcysteamine, 1.0 mM dibutyryl cAMP, 10 ng/ml epidermal growth factor(EGF) and 25% (v/v) porcine follicular fluid (pFF prepared bycentrifugation (2,000×g for 15 min) of the material collected fromantral follicles (3 to 6 mm in diameter), stored at −20° C. and filteredthrough a sterile 0.22 μm pore filter (Millipore, MA, USA) immediatelyprior to use)

[0110] Dulbecco's Phosphate Buffered Saline (DPBS; 136.98 mM NaCl, 2.68mM KCl, 0.49 mM MgCl₂.6H₂O, 8.08 mM Na₂HPO₄, 1.47 mM KH₂PO₄ and 0.90 mMCaCl₂.2H₂O; pH 7.4) supplemented with 1% Foetal Calf Serum (FCS))

[0111] Hepes buffered MEM, consisted of Minimum Essential Medium (withEarle's salts, L-glutamine and non-essential amino acids; Gibco-BRL,Grand Island, N.Y., USA) supplemented with 336 mg/L NaHCO₃, 21 mM Hepesbuffer, 60 mg/L penicillin-G and 0.5% Bovine Serum Albumin (BSA)

[0112] pNCSU-23 (127.8 mM NaCl, 4.97 mM KCl, 1.0 mM KH₂PO₄, 1.19 mMMgSO₄.7H₂O, 3.0 mM Na₂HPO₄, 5.55 mM D-glucose, 75 mg/L penicillin-G, 50mg/L streptomycin sulfate, 1.7 mM CaCl₂, 1.0 mM L-glutamine, 7.0 mMtaurine, 5.0 mM hypotaurine, 0.4% BSA and 10% FCS)

[0113] NCSU-23 (108.73 mM NaCl, 4.78 mM KCl, 1.19 mM KH₂PO₄, 1.19 mMMgSO₄.7H₂O, 5.55 mM D-glucose, 75 mg/L penicillin-G, 50 mg/Lstreptomycin sulfate, 1.7 mM CaCl₂, 1.0 mM L-glutamine, 7.0 mM taurine,5.0 mM hypotaurine and 0.4% BSA)

[0114] Dulbecco's Modified Eagle Medium (DMEM; high glucose withL-glutamine, 110 mg/L sodium pyruvate and pyridoxine hydrochloride:Gibco-BRL)

[0115] Calcium-free mannitol fusion medium (0.28 M mannitol, 0.2 mMMgSO₄ and 0.01% polyvinylalcohol)

[0116] Modified TALP-PVA medium (114.0 mM NaCl, 3.16 mM KCl, 0.35 mMNaH₂PO₄.2H₂O, 0.5 mM MgSO₄.6H₂O, 25 mM NaHCO₃, 2 mg/L phenol red, 0.1%PVA, 75 mg/L penicillin-G, 50 mg/L streptomycin sulfate, 4.72 mMCaCl₂.2H₂O, 10.0 mM sodium lactate, 0.10 mM sodium pyruvate, 2.0 mMcaffeine-sodium benzoate, 3.0 m calcium lactate and 0.4% BSA)

[0117] Methods:

[0118] The preparation of in vitro matured (IVM) oocytes was essentiallyas described previously (Grupen et al., 1997) Reproduction FertilityDevelopment 9: 571-575). Ovaries from slaughtered gilts or, morepreferably, sows were transported to the laboratory in Dulbecco's PBSsupplemented with 0.6% (v/v) of an antibiotic solution (CSL Ltd,Melbourne, Australia) containing penicillin (10,000 U/ml), streptomycin(10,000 μg/ml) and fungizone (25 μg/ml) and maintained at 38° C. Antralfollicles (3 to 6 mm in diameter) were aspirated using a 21-gaugeneedle. The follicular contents were pooled in a collection tube.Cumulus-oocyte complexes (COCs) with at least three uniform layers ofcompact cumulus cells were recovered from the collected fluid, washedthree times in OMM199a, transferred to 50 μl droplets of OMM199a coveredwith mineral oil (≈30 COCs per droplet) and incubated at 38.5° C. in ahumidified atmosphere of 5% CO2 in air. After 22 hr of maturation,expanded COCs were washed once in OMM199a without dibutyryl cAMP(OMM199b), transferred to 50 μl droplets of OMM199b covered with mineraloil and incubated for a further 24 hr. At the end of the 42 h maturationinterval, oocytes were treated with 0.5 mg/ml hyaluronidase for 1 minand gently aspirated with a small bore glass pipette to remove thecumulus cells. Oocytes that had extruded a polar body were washed 3times and cultured in NCSU 23 with 10% FCS at 5% CO₂, 39° C. for 0.5-2 hprior to micromanipulation.

[0119] Primary cultures of porcine foetal fibroblast cells grown toconfluency after 7-14 days culture in DMEM with 15% FCS at 5% CO₂, 39°C. were used as karyoplasts. They were prepared for nuclear transfer bywashing confluent monolayers twice with DPBS followed by the addition ofDPBS containing 0.05% trypsin. After 5 minutes of incubation at 39° C.,DMEM+15% FCS was added to dissociated cells to stop the trypsinreaction. Dissociated cells were then pelleted by centrifugation at300×g for 5 minutes and resuspended in DMEM+15% FCS. Dissociated cellswere incubated at 5% CO₂, 39° C. for at least 0.5 h prior tomicromanipulation.

[0120] For micromanipulation, oocytes and cells were placed in a dropunder oil of pNCSU 23 with 7.5 μg/ml cytochalasin B and 10% FCS. Oocyteswere enucleated by removing the first polar body along with adjacentcytoplasm containing the metaphase plate using a micropipette with aninner diameter of about 20 μm. In a majority of oocytes, the metaphaseplate was visible under phase contrast optics as a clear spacecontrasted against dark cytoplasm. Through the same hole in the zonapellucida created during enucleation, a small to medium-sized cell wasthen placed in contact with the cytoplasm of each oocyte to create acouplet. After manipulation, couplets were washed once and cultured inNCSU 23 with 10% FCS at 39° C., 5% CO₂ in air for at least 0.5 h beforefusion.

[0121] Just prior to fusion, couplets were removed from the incubatorand placed in drops of Ca-free pNCSU 23 under oil for 15 minutes priorto fusion. Groups of up to 10 couplets were washed thoroughly incalcium-free Mannitol fusion medium and then immediately transferred toa fusion chamber with two electrodes 1 mm apart overlaid with fusionmedium. Couplets were manually aligned using a 30 gauge needle so thatthe plane of contact between the donor and recipient cells was parallelwith the electrodes. Cell fusion was induced with a single DC pulse of150 V/mm for 60 μsec. Couplets were also exposed to a 4.0V AC pulse for2 sec immediately prior to the fusion pulse and to an AC pulseimmediately after the fusion pulse, that diminished from 4.0 to 0.0Vover a 2 sec interval. After electrical pulse, couplets were returned toCa-free pNCSU 23 drops for at least 15 min. Unfused couplets wereexposed to the same fusion procedure a second time. Fused embryos werereturned to the incubator at 5% CO₂, 39° C. in NCSU 23 with 10% FCS for1-3 h prior to activation.

[0122] Activation of reconstructed porcine cybrids (fused couplets) wasconducted using the ionomycin/6-DMAP (6-dimethylaminopurine) methoddescribed hereinafter. One to 3 h post fusion, the fused couplets (orsingle cell nuclear transfer embryos) were placed in modified TALP-PVAmedium supplemented with 3.0 mM Ca-lactate (mTALP-PVA) for approximately0.5 hour prior to activation. Fused couplets were then transferred tomTALP-PVA containing 5 μM ionomycin for five minutes. Fused coupletswere then washed twice and incubated in culture medium (NCSU 23+0.4%BSA) containing 2 mM 6-dimethylaminopurine (6-DMAP) for three hours.Activated fused couplets were then washed twice and transferred to 50 μldroplets of the culture medium under mineral oil and cultured for either7 days to assess in vitro development or for 3 days prior to transferinto a synchronised recipient.

[0123] Results:

[0124] The importance of pre- and post-fusion incubation in calcium freepNCSU-23 in avoiding concurrent activation of IVM oocytes isdemonstrated in Table 4. When oocytes were exposed to fusion pulse inCa-free mannitol fusion medium, oocytes pre- and post incubated in Cacontaining pNCSU-23 were found to cleave at a high rate of 93% after 2days of culture indicating that the majority underwent activation.However, only 17% of oocytes cleaved when pre- and post incubated inCa-free pNCSU-23 suggesting that the vast majority remained unactivated.TABLE 4 Development of IVM sow oocytes after exposure to fusion pulsewhen pre- and post-incubated in either Ca-free or Ca- containingpNCSU-23 Treatment Number Cleaved Blastocyst Pre- and post-incubationwith calcium 44 41 (93) 4 (9) Pre- and post-incubation without calcium58 10 (17) 0 (0)

[0125] When couplets were fused in Ca-free mannitol and incubated pre-and post-fusion in Ca-free pNCSU-23, high development to blastocyststage (21%) was achieved after 7 days of culture after they wereactivated (3 h after fusion) using ionomycin/6-DMAP treatment (TABLE4b). When fused couplets were not activated using ionomycin/6-DMAP, only17% cleaved indicating that fusion occurred without concurrentactivation in the majority of cybrids. TABLE 4b Development of NTcybrids using IVM pig oocytes with or without activation usingionomycin/6-DMAP Treatment Number Cleaved (%) Blastocyst (%) NTactivated 105 63 (60) 22 (21) NT not activated 76 13 (17) 0 (0)

[0126] The invention has been described herein, with reference tocertain preferred embodiments, in order to enable the reader to practicethe invention without undue experimentation. However, a person havinggeneral skill in the art will readily recognise that many of thecomponents and parameters may be varied or modified to a certain extentwithout departing from the scope of the invention. Furthermore, titles,headings, or the like are provided to enhance the reader's comprehensionof this document, and should not be read as limiting the scope of thepresent invention.

[0127] The entire disclosures of all applications, patents andpublications, cited above and below, if any, are hereby incorporated byreference.

[0128] The reference to any prior art in this specification is not, andshould not be taken as, an acknowledgment or any form of suggestion thatthat prior art forms part of the common general knowledge in the fieldof endeavour relevant to the subject matter of this specification.

[0129] Throughout this specification, unless the context requiresotherwise, the word “comprise”, and variations such as “comprises” and“comprising”, will be understood to imply the inclusion of a statedinteger or step or group of integers or steps but not the exclusion ofany other integer or step or group of integers or steps.

1. A method for the production of nuclear transfer embryos comprising atleast the steps of: providing at least one enucleated recipient cell;providing at least one donor cell or nucleus; placing said at least oneenucleated recipient cell and at least one donor cell or nucleus incontact with one another to form couplets; providing a fusion mediawhich is substantially free of calcium; and fusing via electrofusion, insaid fusion media, said at least one recipient cell with at least onedonor cell or nucleus to form a nuclear transfer embryo.
 2. A method asclaimed in claim 1 wherein said recipient and donor cells are porcine.3. A method as claimed in claims 1 or 2 wherein the parameters of saidelectrofusion are a single electrical pulse at between 100V to 200V forbetween 30 μs and 100 μs over an electrode gap of 1 mm.
 4. A method asclaimed in claims 1 or 2 wherein the parameters of said electrofusionare a single electrical pulse at 1.5 kV/cm for 60 μs.
 5. A method asclaimed in any one of claims 1 to 4 wherein said recipient cell is afreshly ovulated or a follicular oocyte arrested at MII.
 6. A method asclaimed in any one of claims 1 to 4 wherein said recipient cell is an invitro-matured oocyte.
 7. A method as claimed in any one of claims 1 to 6wherein the couplets are held in media which is substantially free ofcalcium for a period prior to electrofusion to form a nuclear transferembryo.
 8. A method as claimed in claim 7 wherein the period is at leastapproximately 15 minutes.
 9. A method as claimed in any one of claims 1to 8 further comprising the step of incubation in a media which issubstantially free of calcium for a period following electrofusion. 10.A method as claimed in claim 9 wherein the period is at leastapproximately 15 minutes.
 11. A method as claimed in any one of claims 1to 10 wherein said donor cell or nucleus is a somatic cell.
 12. A methodas claimed in claim 11 wherein said donor cell is a fibroblast.
 13. Amethod of cloning animals comprising at least the steps of: producing anuclear transfer embryo according to a method as claimed in any one ofclaims 1 to 12; activating said nuclear transfer embryo to provide anactivated embryo; optionally allowing said activated embryo to undergoat least one round of cell division; transferring activated and dividedembryo to a synchronised female recipient animal; allowing saidsynchronised female recipient animal to carry said embryo to fullgestation to produce a cloned animal.
 14. A method as claimed in claim13 wherein said NT embryo is porcine and said recipient female animaland said cloned animal are pigs.
 15. A method as claimed in claims 13 or14 wherein where the nuclear transfer embryo is constructed using invivo-derived oocytes, said nuclear transfer embryo is held in acalcium-containing media with serum prior to activation thereof.
 16. Amethod as claimed in claims 13 or 14 wherein where the nuclear transferembryo is constructed using in vitro-matured oocytes, said nucleartransfer embryo is held in a media substantially free of calcium for aperiod prior to activation thereof.
 17. A method as claimed in claim 16wherein the period is at least approximately 15 minutes.
 18. A method asclaimed in any one of claims 13 to 17 wherein said nuclear transferembryos are activated no later than 5 hours post fusion.
 19. A methodfor the production of porcine nuclear transfer embryos comprising atleast the steps of: providing at least one enucleated recipient cell ofporcine origin; providing at least one donor cell or nucleus of porcineorigin; placing said at least one enucleated recipient cell and at leastone donor cell or nucleus in contact with one another to form couplets;optionally providing a first media which is substantially free ofcalcium; optionally incubating said couplets in said first media for aperiod of preferably at least approximately 15 minutes; providing asecond media which is substantially free of calcium; fusing viaelectrofusion, in said second media, said at least one recipient cellwith at least one donor cell or nucleus to form a nuclear transferembryo; providing a third media which is substantially free of calcium;and incubating said nuclear transfer embryo in said third media for aperiod preferably of at least approximately 15 minutes followingelectrofusion.
 20. A method of cloning pigs comprising at least thesteps of: producing a porcine nuclear transfer embryo according to claim19; activating said nuclear transfer embryo to provide an activatedembryo; optionally allowing said activated embryo to undergo at leastone round of cell division; transferring activated and divided embryo toa synchronised female recipient animal; allowing said synchronisedfemale recipient animal to carry said embryo to full gestation toproduce a cloned pig.